The present invention relates to methods of treating gout with inhibitors of the nuclear enzyme poly(adenosine 5xe2x80x2-diphospho-ribose) polymerase [xe2x80x9cpoly(ADP-ribose) polymerasexe2x80x9d or xe2x80x9cPARPxe2x80x9d, which is also referred to as ADPRT (NAD:protein (ADP-ribosyl transferase (polymersing)) and PARS (poly(ADP-ribose) synthetase) and provides compounds and compositions containing the disclosed compounds for use in the disclosed method.
Reviews of PARP as well as the effects of inhibiting the same may be found, for example, in PCT/US98/18184, PCT/US98/18226, PCT/US98/18187, PCT/US98/18195, PCT/US98/18196, PCT/US98/18188, PCT/US98/18189, PCT/US98/18185, PCT/US98/18186, the entire contents of each of which are hereby incorporated by reference.
Deposition of crystals of monosodium urate (MSU crystals) in the joint articular space is the etiological cause of inflammatory pathologies such as gout and pseudogout. Clinically, these inflammatory diseases are associated with oedema and erythema of the joints with consequently severe pain. A strong infiltration of leucocytes in the intraarticular and periarticular space leading to: 1) acute, episodic articular and periarticular inflammation, and 2) chronic articular changes, are also characteristic of this pathology. It has long been clear that neutrophils are the predominant cell type recovered from these inflammatory joints (Dieppe et al., (1979). Synovial fluid crystals. Q. J. Med. XLVIII: 533-553; Terkletaub, (1991). Monocyte-derived neutrophil chemotactic factor/interleukin-8 is a potential mediator of crystal-induced inflammation. Arth. Rheum. 34: 894-903.). A better understanding of the inflammatory processes elicited by MSU crystals, and the fact that there is a clear relationship between these crystals and gouty arthritis, has prompted the characterisation of experimental models of crystal-induced inflammation. Examples of models where crystal challenge has led to call recruitment into specific cavities, are canine joints (Phelps and McCarty, 1966, Ann Int. Med. 9: 115-125), rat pleurisy (Deporter et al., 1979, Br. J. Pharmacol. 65: 163-165; Sedgwick et al., 1985, Agents Actions 17: 209-213), and utilisation of a pre-formed rat air-pouch (Brookes et al., 1987). The latter experimental system has shown that neutrophil accumulation was related to generation of chemoattractants such as LTB4, which was subsequently inhibited by colchicine (Brooks et al., 1987, Br. J. Pharmacol. 90: 413-419).
Neutrophils have been shown to be activated by MSU crystals, releasing an array of mediators that may be, in part, responsible for the local and systemic inflammatory manifestations found in crystal-induced joint disorders. The crystals interact with neutrophils leading to the release of lysomal enzymes (Hoffstein et al., 1975, Arth. Rheum. 18: 153-165), release of oxygen derived free radicals (Simchowitz et al., 1982, Arth. Rheum. 25: 181-188; Abramson et al., 1982, Arthr Rheum. 25: 174-180), induction of phospholipase A2 (PLA2) in leucocytes (Bomalaski et al., 1990, J. Immunol. 145: 339:-3397), and activation of synthesis of 5-lipoxygenase products (Poubelle et al., 1987, Biochem. Biophys. Res. Commun. 149: 649-657).
In vitro, MSU crystals have been shown to release the cytokine interleukin-1xcex2 (IL-1xcex2) from human neutrophils, adding this stimulus to a list of others that also release this cytokine, such as zymosan, LPS, phorbol esters, granulocyte macrophage-colony stimulating hormone (GM-CSF) and TNF-alpha. Furthermore it has also been shown that human monocytes and synoviocytes can synthesise and release various cytokines such as IL-6 and IL-8 (Guerne et al., 1989, Arth. Rheum. 32: 1443-1452; Terkeltaub et al., 1991, Arth. Rheum. 34: 894-903). In addition, colchicine selectively inhibits MSU crystal- and TNF-Z induced release of IL-1xcex2 (Roberge et al., 1994, J. Immunol. 152: 5485-5494).
In experimental models of gout the synthesis of a CXC chemokine selective for neutrophils, such as IL-8, has also been observed, but not that of a CC chemokine monocyte chemoattractant protein-1 (MCP-1) (Hachicha et al., 1995, J. Exp. Med. 182: 2019-2025). These results suggest that production of IL-8 and abolition of the release of MCP-1, will lead to an event where, theoretically there will be a recruitment of neutrophils but not mononuclear cells. This hypothesis is in accordance with the pathological state of gout and pseudogout, where the predominant inflammatory cell is the neutrophil (Hachicha et al., 1995). In addition MSU crystal activation of mononuclear phagocytes, which are normally found in the joint space, also induces secretion of IL-8 (Terkeltaub et al., 1991). The importance of IL-8 in this pathology has been shown in synovial fluids of patients with acute gouty arthritis where it occurs in elevated amounts (Terkeltaub et al., 1991; di Giovine et al., 1991, J. Clin. Invest. 87: 1375-1381). The use of a neutralising antibody against IL-8 has been shown significantly to attenuate the crystal induced joint swelling at 12 h and neutrophil infiltration into arthritic joints at 12 and 24 h in a rabbit model (Nishimura et al., 1997, J. Leukoc. Biol. 62: 444-449).
These studies demonstrate the importance of both the emigrating neutrophil and the chemokine IL-8, as well as the release of this and other cytokines from resident cells such as the synoviocytes, macrophages and mast cells in treating gout. Since neutrophils are not present or are extremely rare in normal synovial fluid, enhanced neutrophil-endothelial adhesion is necessary for gout to occur (Terkeltaub, 1996, In. Koopman, W. J. editor. Arthritis and allied conditions: a textbook of rheumatology. Baltimore: Williams and Wilkins: pp. 2085-2102, and Terkeltaub, 1992, In Inflammation. Basic Principles and Clinical Correlates, ed. by J. I. Gallin, I. M. Goldstein and R. Snyderman, pp 977-981, Raven Press, New York). IL-1xcex2 and TNF-alpha may be critical in mediating the rapid up-regulation of the major endothelial ligand for neutrophils. For instance rapid and prolonged expression of E-selectin in response to injection of urate crystals has been demonstrated in pig skin (Chapman et al., 1996, Br. J. Rheumatol. 35: 323-334). The release of cytokines, chemokines and products of the arachidonic acid cascade system lead to the recruitment of neutrophils in this pathology, and inhibition of these leads to an attenuation of the pathology.
The following gout model was used to test a PARP inhibitor according to the present invention.
Male outbread Swiss albino mice (20-22 g body weight) were purchased from Banton and Kingsman (T.O. strain; Hull, Humberside) and maintained on a standard chow pellet diet with tap water ad libitum and a 12:00 h light/dark cycle. All animals were housed for 1 week prior to experimentation to allow body weight to reach 28-30 g. 1,11b-dihydrobenzopyrano[4,3,2-de ]isoquinolin-1-one was dissolved in 100% DMSO at room temperature at a concentration of 45 mg in 2 ml. The compound was then injected into the peritoneal cavity, so as each mouse received a single dose corresponding to 45 mg/2 ml/kg (e.g. 60 xcexcl for a mouse of 30 g). Control mice received DMSO at 2 ml/kg i.p. A third group of mice which were left untreated were added to control for potential effects of the vehicle. The study involved therefore, the following three groups: group A, untreated mice, n=6, group B, DMSO-treated mice, n=8, and group C, mice treated with 1,11b-dihydrobenzopyrano[4,3,2-de ]isoquinolin-1-one, n=8
MSU crystal-induced neutrophil recruitment was tested as follows. In all cases, mice were treated 1 h after the treatment noted above, with MSU crystals. A homogenous suspension of MSU crystals was obtained by a 30 min rotation. Peritonitis was induced by injection of 3 mg MSU crystals in 0.5 ml PBS (0.1 M, pH 7.4), and the recruitment of neutrophils into the cavity evaluated at the 6 h time point (Getting et al., 1997, J. Pharmacol. Exp. Ther. 2.33: 123-130). Animals were then euthanised by CO2 exposure and the peritoneal cavity washed with 3 ml of PBS supplemented with 3 mM EDTA and 25 U/ml heparin.
An aliquot (100 xcexcl) of the lavage fluid was then diluted 1:10 in Turk""s solution (0.01% crystal violet in 3% acetic acid). The samples were then vortexed and 10 xcexcl of the stained cell solution were placed in a Neubauer haematocymometer and neutrophils numbers counted using a light microscope (Olympus B061). Cell-free supernatants have been prepared by centrifugation and stored for potential future analysis.
Data are shown for single mice, and also shown as meanxc2x1S.E. of (n) mice per group. Statistical differences were determined by ANOVA, plus Bonferrconi test. A P value  less than 0.05 was taken as significant.